Saturday , July 4 2020




Section No Topics Page No
1.0 Protocol Approval
2.0 Objective
3.0 Acceptance Criteria
4.0 Responsibilities
5.0 Validation Methodologies
6.0 Evaluation of test Result
7.0 Re-validation Frequency
8.0 Documentation
9.0 Conclusion
 Name /  Designation  Signature  Date
 Prepared By
 Checked By
Manager. Quality Control
 Approved By
 Plant Manager    
Manager. Quality Assurance  


To evaluate the efficiency of disinfectants which are being used for the surface and area sanitization of controlled and critical clean rooms.


  • The disinfectants when used as per the recommendations of the manufacture for  dilution and the contact time should be able to inhibit the growth of the test microorganisms .


Validation Group consists of following members :

  • QA/QC Department Representative

Manager, QA

Manager QC

Manager, Microbiology


  • Specific Responsibilities
    • Microbiologist will write the protocol in consultation with QC Manager .
    • Manager, QA will check the protocol for its completeness, accuracy, technical excellence and applicability.
    • Head of the Department QA will be responsible for final approval of protocol.
    • Microbiologist will be responsible for preparation of slants , sub culturing of organisms , streaking on the slants .
    • Microbiologist will also be responsible for preparing the used dilution as per the manufacturers recommendation.


Before proceeding for validation following materials are required.

Staphylococcus aureus



Candida albicans

Apergillus niger.

Micrococcus species Environmental Isolate

  • Disinfectants for Validation.
  • Sterile distilled water.
  • Sterile Molten Soyabean Caesin Digest Agar
  • Sterile Molten Potato Dextrose Agar
  • Poured SCDA plates.
  • Poured PDA plates
  • Sterile forceps
  • Sterile membrane filtration units.
  • Sterile membranes
  • Vortex Mixer.

I  –   Preparation of Spore forming Culture

  1. Prepare SCDA slants as per the SOP.
  2. Incubate slant for 48 hrs at 30 – 35 ° C as pre-incubation to cross check the contamination.
  3. from the working culture streak a loopful of Bacillus subtilis culture on to the newly prepared slant.
  4. Incubate the slants at 30 – 35 ° C for 48 hrs.
  5. After 48 hrs of incubation cross check the culture for any cross contamination by simple gram staining technique.
  6. During Gram Staining also check for the presence of vegetative or spore cells.
  7. After Gram Staining preserve the cultures for further 7 days.
  8. After 7 days cross check the culture for any spore formation by simple Negative Staining technique.

 II – Negative staining.

  1. Prepare a thin smear on a clean slide.
  2. Place the slide on a staining rack above boiling water.over the smear with small pieces of tissue paper.
  3. Pour malachite green and continue heating till boiling for 5 minutes.
  4. Do not allow the stain to dry.
  5. Gently wash the slide with water.

III-  Preparation of Challenge Inoculam.

  1. Prepare fresh SCDA /PDA slants as per the SOP 12043.
  2. Incubate those slants at 30 – 35 ° C for 48 hrs to cross check any type of contamination.
  3. From the working culture streak a loopful of culture into the freshly prepared slants as per the SOP.
  4. Incubate those inoculated slants at 30 – 35 ° C for 48 hrs for bacterial cultures and 20 – 25 ° C for 5 days for fungal cultures.
  5. Add 5.0 ml of sterile saline into the slants aseptically taking care not to contaminate the slants .
  6. With the help of a sterile spatula streak the lawn of organisms.
  7. Take 1.0 ml of the culture suspension and make serial dilutions ranging from 1:10 to 1: 100 .
  8. Plate the serial dilutions from 1:10  to 1:100 taking 1.0 ml of the culture in sterile petri-dishes.
  9. Pour sterile molten  SCDA and PDA on to the inoculated plates.
  10. Incubate the plates at the required specified temperature.
  11. After plating the required dilutions do not discard the dilutions and preserve the samples at a temperature of 2 – 8  ° C till the incubation period .
  12. After the incubation count the number of colonies .
  13. Select the dilution which is having 10000 to 100000 cells / ml for validation study .
  14. Preserve the culture suspension.Record the data in Annexure. 

IV- Preparation of Disinfectant solution

  • Take the concentrated solution as received from the supplier and dilute the disinfectant to prepare a stock solution, which is twice that of the recommended concentration.
  • This will be the used dilution.

V –   Determining the initial microbial count.

  1. With the help of an calibrated micropipette pipette out 1.0 ml of any of the culture and inoculate into 9.0 ml of the 0.9 % sterile saline solution.
  2. Vortex it for 5.0 minutes.
  3. Filter the sample through a 0.45 m membrane.
  4. Give three washings of 100 ml each with .1 % sterile peptone water.
  5. After filtration , with the help of a sterile forcep take the membrane and place the membrane  on a SCDA or m (HPC) agar plate.
  6. Incubate the plate .
  7. After incubation count the number of colonies present on the membrane.
  8. Note down the number of colonies.
  9. This will be the initial counts of the culture.
  10. In the same manner proceed for all the other cultures which are going to be tested.
  11. Record the initial counts in the Annexure and report as Counts / 0.1 ml

IV – Determining the Efficacy of the Disinfectant by Use Dilution Method.

  1. Prepare test tubes having 9.0 ml of sterile distilled water.
  2. Add 1.0 ml of the use dilution for one disinfectant.
  3. Vortex the tube for 5.0 minutes.
  4. Add 0.1 ml of any one culture into the test sample.
  5. Make the sample dilution in such a way that each contact time have two sets of samples.
  6. Give a contact time of 0 hrs, 5.0 min , 10 min .
  7. After the specified  contact time,  filter the samples through a 0.45 m  membrane.
  8. Give three washings of 100 ml each with .1 % sterile peptone water.
  9. After filtration with  the help of a sterile forcep take the membrane and place it on a SCDA or m (HPC) agar.
  10. Incubate the plates .
  11. After incubation count the number of colonies present on the membrane.
  12. Note down the number of colonies.
  13. This will be the final counts of the exposed culture .
  14. Select the plates which have least to Nil counts.
  15. Proceed in the same manner taking all the cultures to be tested .
  16. Contact time for the usage of the disinfectant will be set on the basis of the results which will have least counts. 

V- Determining the Efficacy of the Disinfectant by Surface Method

  1.  To get a practical approach for the efficacy  apply the disinfectants on to any of the surface which is present in that particular area which will be  decontaminated by spraying the disinfectant.
  2.  Take S.S strips and Epoxy coated material having a surface area of 25 cm2.
  3.  Wrap the strips and the epoxy coated surface with a aluminium foil and sterilize it in an Dry heat Steriliser at 200 0  C for 2 hrs.
  4. From the previously determined suspension having 10000 – 100000 cells per ml inoculate one culture on to three different S. S surface and Epoxy coated surface.
  5. With the help of a sterile spatula  spread the culture on the surface.
  6. Keep it on the LAF bench for 30 minutes for drying.
  7. After the exposed duration for drying spray the disinfectant on to any one  surface of the recommended used dilution .
  8. Allow the surface to be with the sanitiser for 0hrs, 5.0 min , 10 min.
  9. With the help of a sterile moistened swab , swab the surface gently covering all the area of the surface.
  10. Use different swabs for all the strips.
  11. Place the two swab sticks in a test tube having sterile solution of fluid caesin digest Soya broth .
  12. Vortex the test tube gently for 5. 0 minutes .
  13. Aseptically filter the samples through a 0.45 m  membrane.
  14. Give three washings of 100 ml each with .1 % sterile peptone water.
  15. After the filtration with  the help of a sterile forcep take the membrane 5.52  and place it on a SCDA or m (HPC) agar.
  16. Incubate the plates .
  17. After incubation count the number of colonies present on the membrane.
  18. Note down the number of colonies in both the test sample and that with the unexposed strip.
  19. Proceed in the same manner taking all the cultures to be tested and the various sanitisers. 


The decrease in the bacterial load to the exposed disinfectant indicates that the disinfectant is capable of reducing the contaminant when used in the  area.

7.0       Schedule Revalidation:

Revalidation shall be carried out in case of

  • A new disinfectant is received .
  • If the manufacturer revise the concentration of ingredients.
  • If a new microorganism is isolated from that particular area. 


Validation Report contains the following documents.

  • Approved Validation protocol
  • Media Preparation as per SOP.
  • Gram Staining GTP.
  • Test report for different disinfectants
  • List of Approved Disinfectants.
  • Summary Conclusion .

9.0       CONCLUSION :

Summary report will contain discussion and conclusion which clearly state the successful achievement of objective of validation studies.

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About Pharmaceutical Guidanace

Mr. Shiv Kumar is the Author and founder of pharmaceutical guidance, he is a pharmaceutical Professional from India having more than 14 years of rich experience in pharmaceutical field. During his career, he work in quality assurance department with multinational company’s i.e Zydus Cadila Ltd, Unichem Laboratories Ltd, Indoco remedies Ltd, Panacea Biotec Ltd, Nectar life Science Ltd. During his experience, he face may regulatory Audit i.e. USFDA, MHRA, ANVISA, MCC, TGA, EU –GMP, WHO –Geneva, ISO 9001-2008 and many ROW Regularities Audit i.e.Uganda,Kenya, Tanzania, Zimbabwe. He is currently leading a regulatory pharmaceutical company as a head Quality. You can join him by Email, Facebook, Google+, Twitter and YouTube

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